All events in vivo are caused directly or indirectly by changes of intracellular gene actions, and thus, it has been positioned as an important analysis method to measure an activity of gene transcription which occurs intracellularly in the field of life science. One of the analysis methods is referred to as a reporter assay, which is the method in which a reporter enzyme gene is inserted downstream of a gene sequence (gene transcription regulatory region) controlling a gene action, and an amount of a reporter enzyme which changes along with the change of the gene transcription regulatory region is evaluated by its enzyme activity, thereby evaluating the amount of gene transcription.
The most popular one as the reporter enzyme is firefly luciferase, and the transcription activity is evaluated by introducing a vector in which a firefly luciferase gene has been inserted downstream of the gene transcription regulatory region into cells and measuring an amount of luminescence which is the firefly luciferase enzyme activity expressed in the cells. In this measurement, when only one reporter enzyme is available, it is difficult to relatively evaluate a measured value because only one gene activity is measured. Thus, a dual reporter analysis by comparing with the value of transcription activity in the gene transcription regulatory region as the control has been developed, and a control reporter gene obtained by inserting an SV40 or CMV promoter sequence upstream of a Renilla luciferase gene taking a different luciferin (substrate) structure has been used. In this method, two gene transcription activities are evaluated by adding each luciferin to a cell lysate and measuring each amount of the luminescence, and the method has been commercialized by Promega. The other method in which expression amounts of three genes are evaluated in a multiple gene expression detection system similarly using firefly luciferin but using green, orange and red luciferases derived from beetles having different luminescent colors has been in practical use (Nakajima et al, Biotechniques, vol. 38, 891-894). In this method, three gene transcription activities are evaluated by adding firefly luciferin into the cell lysate and separating and quantifying mixed luminescence spectra, and the method has been commercialized by Toyobo.
In the reporter assay, the cells are stimulated for a certain time period, and the change of an expression amount of a particular gene is evaluated as an accumulated amount of the reporter enzyme in the cells. Thus, the accumulated amount is measured by lysing the cells which has passed over the certain time period. Therefore, it is impossible to measure with time using the same cell group or measure the same cell group again. In order to compensate this shortcoming, secretory luciferase has been noticed, and luciferases using Cypridina luciferase (JP 2004-187652-A) and copepod luciferase (Gaussia luciferase) (U.S. Pat. No. 6,436,682) (Prolume) have been in practical use. A secretory luciferase protein expressed by its gene is secreted extracellularly and accumulated in a culture medium. Thus, the expression amount of the gene is evaluated by collecting a part of the culture medium and adding a Cypridina luciferin or coelenterazine solution thereto to measure the enzyme activity of the reporter enzyme. When secretory luciferase is used, it is not necessary to lyse the cells. Thus, there is advantages in that the reporter assay can be performed using the alive cells and the assay with high throughput is possible by the use of a dispenser for a 96-well or 384-well plate. However, it is problematic in that the measured values can not be relatively evaluated because only one information is obtained.
Additionally, a background luminescence of coelenterazine is known to affect the luminescence of copepod luciferase (Anal. Chem. 2002, 74, 4378-4385).